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Background: Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored. Methods: We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7 CreER/+ ; ROSA26 mTmG/+ mice. SCs were depleted in Pax7 CreER/+ ; ROSA26 DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted. Results: Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice. Conclusion: The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals.

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc.

The olfactory epithelium maintains stem and progenitor cells that support the neuroepithelium’s life-long capacity to reconstitute after injury. However, the identity of the stem cells – and their regulation – remain poorly defined. The transcription factors Pax6 and Sox2 are characteristic of stem cells in many tissues, including the brain. Therefore, we assessed the expression of Pax6 and Sox2 in normal olfactory epithelium and during epithelial regeneration after methyl bromide lesion or olfactory bulbectomy. Sox2 is found in multiple kinds of cells in normal epithelium, including sustentacular cells, horizontal basal cells, and some globose basal cells. Pax6 is co-expressed with Sox2 in all these, but is also found in duct/gland cells as well as olfactory neurons that innervate necklace glomeruli. Most of the Sox2/Pax6-positive globose basal cells are actively cycling, but some express the cyclin-dependent kinase inhibitor p27Kip1, and are presumably mitotically quiescent. Among globose basal cells, Sox2 and Pax6 are co-expressed by putatively multipotent progenitors (labeled by neither anti-Mash1 nor anti-Neurog1) and neuron-committed transit amplifying cells (which express Mash1). However, Sox2 and Pax6 are expressed by only a minority of immediate neuronal precursors (Neurog1- and NeuroD1-expressing). The assignment of Sox2 and Pax6 to these categories of globose basal cells is confirmed by a temporal analysis of transcription factor expression during the recovery of the epithelium from methyl bromide-induced injury. Each of the Sox2/Pax6-colabeled cell types is at a remove from the birth of neurons; thus, suppressing their differentiation may be among the roles of Sox2/Pax6 in the olfactory epithelium.

Aims Recently, an association of Type 2 diabetes (T2DM) with polymorphisms in PTPN1 located on chromosome 20q was reported. We attempted to replicate this finding in an ethnically homogeneous Polish population. Methods The study groups comprised 474 cases with T2DM and 411 control subjects with normal fasting glucose. All individuals were genotyped for the five previously reported PTPN1 polymorphisms using a fluorescence polarization method. HAPLO.STAT software was used to infer and compare haplotype distributions. Results The distributions of alleles and genotypes for the five genotyped PTPN1 polymorphisms did not differ between the T2DM cases and control subjects (lowest P = 0.6). Similarly, the frequency of the common haplotype reported to be associated with T2DM did not differ in cases and control subjects. We also failed to find such an association in Whites by performing a meta-analysis of all the available data on the association of those five SNPs with T2DM. Conclusion This case-control study in a Polish population did not confirm the reported association between polymorphisms in PTPN1 and T2DM.

The product of the PPP1R3B gene (G(L)) is the regulatory subunit of PP1 - a serine/threonine phosphatase involved in the modulation of glycogen synthesis in the liver and skeletal muscle. The PPP1R3B gene is located on chromosome 8p23 in a region that has been linked with type 2 diabetes and maturity-onset diabetes of the young (MODY). We examined whether sequence variants at the PPP1R3B locus are responsible for the linkage with diabetes observed at this location. RT-PCR analysis revealed the existence of two alternative promoters. These and the two exons of this gene were sequenced in the probands of 13 Joslin families showing the strongest evidence of linkage at 8p23. A total of 20 variants were observed: two in the 5' flanking region, one in the intron (9 bp 5' of exon 2), and 17 in the 3' UTR. The intronic variant generated a new acceptor splice site, resulting in an alternative splice variant with a longer 5' UTR. However, neither this nor other variants segregated with diabetes in the 13 'linked' families. Furthermore, allele frequencies were similar in 90 family probands from the Joslin Study and 347 unrelated controls. Thus, genetic variability in the PPP1R3B gene does not appear to contribute to diabetes in our mostly Caucasian families. However, a role cannot be excluded in other populations such as the Japanese, among whom linkage to diabetes is also observed at 8p23 and a non-synonymous mutation has been detected in the PPP1R3B gene.

We recently demonstrated that IFNaR2, a subunit of the interferon receptor, can be proteolytically cleaved in response to interferon-alpha and other activators of protein kinase C. Cleavage occurs at multiple sites, via a mechanism similar to that employed by Notch and the Alzheimer's precursor protein, and releases the intracellular domain (ICD). In this study, we demonstrate that the IFNaR2 ICD, when fused to the yeast Gal4 DNA binding domain (Gal4DBD) selectively modulates transcription of four different promoters under the control of Gal4 upstream activating sequences. We previously showed that Stat2 binds constitutively to the ICD of IFNaR2, in a manner that is independent of tyrosine phosphorylation. Here, we show that lCD transcriptional modulation is dependent upon the carboxyl-terminal transactivation domain of Stat2. Specifically, complementing Stat2 deficient cells with wildtype Stat2 restored the ICD-mediated transcriptional effects while complementation with a mutant form of Stat2 lacking the transcriptional activation domain (TAD) did not. In addition, mutation of the Stat2 binding site on the ICD reduced the transcriptional activity of the Gal4DBD-ICD. Finally, we demonstrate that the activity of jak1, a tyrosine kinase also known to bind to IFNaR2, is required for ICD-mediated transcriptional effects. J. Cell. Physiol. 204: 567-573, 2005. (c) 2005 Wiley-Liss, Inc.

Summary Aims To investigate association and linkage between DNA sequence variants in the aldose reductase (AR) gene on chromosome 7q35 and diabetic nephropathy (DN) in Type 1 diabetes mellitus. Methods By sequencing the promoter region and 10 exons in eight DN cases and eight controls, a frequent bi-allelic polymorphism (C-106T) was discovered. This polymorphism and the known 5′ALR2 dinucleotide repeat polymorphism were genotyped in unrelated cases with advanced nephropathy (n = 221) and unrelated controls with normoalbuminuria (n = 193). For a family based study, 166 case-trios (case and both parents) and 83 control-trios (control and both parents) were also genotyped. Results In the case–control study, carriers of the Z-2 allele of the 5′ALR2 polymorphism had a significantly higher risk of DN than non-carriers (odds ratios: 1.6 for heterozygotes and 2.1 for homozygotes, P

In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21(waf1/cip1) (p21), p27(kip1) (p27), and p16(ink4a) (p16) and/or the positive regulators cyclin D(1) and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T(1/2) cell clones transformed by 1.5 Gy of alpha radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following gamma irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D(1) was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones.

BACKGROUND. Families with a high incidence of hereditary breast cancer, and subsequently shown to have terminating mutations in BRCA1 or BRCA2, appear to have a higher incidence of prostate cancer among male relatives. We aimed to determine whether the common germline mutations of BRCA1 or BRCA2 in Ashkenazi Jewish men predisposed them to prostate cancer. METHODS. We examined genomic DNA from 83 (for BRCA1 185delAG) or 82 (for BRCA2 6174delT) Ashkenazi Jewish prostate cancer patients, most of whom were treated at a relatively young age, for the most common germline mutation in each gene seen in the Ashkenazi population. RESULTS. Our study should have been able to detect a 4‐5-fold increase in the risk of prostate cancer due to mutation of BRCA1 or BRCA2. However, only one (1.15%; 95% confidence interval, 0‐3.6%) of the patients was heterozygous for the BRCA1 mutant allele, and only two were heterozygous for the BRCA2 mutation (2.4%; 95% confidence interval, 0‐6.2%). CONCLUSIONS. The incidence of each of the germline mutations in these prostate cancer patients closely matched their incidence (about 1%) in the general Ashkenazi Jewish population. This suggests that unlike cases of breast and ovarian cancers, mutations in BRCA1 or BRCA2 do not significantly predispose men to prostate cancer. Prostate 40:172‐177, 1999. © 1999 Wiley-Liss, Inc.

Summary Aims Mutations in hepatocyte nuclear factor (HNF)-4α gene located on chromosome 20q have been found to be responsible for the development of early onset Type 2 diabetes mellitus (DM). Through a national campaign, 53 families with autosomal dominant, early onset Type 2 DM (n = 654) were assembled to determine the frequency of mutations in the HNF-4α gene and their contribution to the development of diabetes. Methods Twelve exons and the promoter region of the HNF-4α gene were screened in probands of the families by a double gradient, denaturing gradient gel electrophoresis (DG-DGGE) protocol combined with automated bi-directional sequencing of the PCR products of all heterozygous individuals. Results We detected two new mutations in the HNF-4α gene that changed the amino-acid sequence. The first mutation was a GlySer substitution in codon 115 within a highly conserved DNA binding domain, and all six carriers of this mutation had diabetes and low insulin secretion. The second mutation was an IleVal substitution in codon 454 within the transactivation domain. It was carried by four family members, two of whom also carried a mutation in the HNF-1α gene. Of those having only the mutation in HNF-4α one had diabetes and the other had normal glucose tolerance and both were obese and hyperinsulinaemic. Thus, it is uncertain that this mutation is responsible for any of the diabetes in this family. Conclusion We have found that mutations in the HNF-4α gene account for a small proportion, about 2–4%, of families with early onset, autosomal dominant, Type 2 DM in US Caucasians. Diabet. Med. 16, 193–200 (1999)

Cowden disease, a dominantly inherited syndrome characterized by a variety of proliferative lesions and predisposition to breast and thyroid cancer, has recently been linked to the polymorphic marker D10S215 on chromosome segment 10q23. Loss of heterozygosity in prostate cancer is linked to the same marker, whereas loss of heterozygosity in glioblastoma, endometrial cancer, and other malignancies also localizes to this region. Most recently, a putative tumor suppressor gene (PTEN/MMAC1) has been identified in the region between D10S215 and an adjacent, more telomeric marker (D10S541) and was found to be altered in breast cancers, prostate cancers, and glioblastomas. We examined 22 invasive breast cancers for loss of heterozygosity in the 10q23 region and found loss in 41% (9/22). There were two distinct regions of loss, including one near the D10S541 marker, with an approximately equal frequency of deletion in each. The observed pattern of deletion is consistent with the presence of a tumor suppressor gene between D10S215 and D10S541. Most of the poorly differentiated carcinomas in the case collection showed loss of heterozygosity in the region near D10S215, suggesting that this loss correlates with a poor prognosis.

Signaling by interferon alpha (IFN alpha), an extracellular factor that mediates a number of anti-viral and growth-suppressive effects, requires two members of the Janus family of tyrosine kinases (JAK family): Jak1 and Tyk2. IFN alpha treatment of cells induces the rapid tyrosine phosphorylation of these two kinases, two subunits of the IFN alpha receptor, and two members of the signal transducer and activator of transcription (STAT) family of latent transcription factors. These proteins are believed to be direct substrates of one or both JAKs. Though the requirement for both Jak1 and Tyk2 in the IFN alpha-signaling cascade is well established, the order of activation and the relative contribution of the two kinases has not been elucidated completely. To address these questions, we have employed kinase-deficient mutants of both enzymes. Both mutant kinases suppress transcriptional activation as measured by an IFN alpha-dependent reporter-gene assay. Furthermore, in transient-transfection assays, the kinase-deficient versions of Tyk2 and Jak1 can act independently to inhibit STAT phosphorylation. Thus, kinase-deficient versions of JAK can act in a dominant-negative fashion to suppress IFN alpha signaling. The effects of the overexpressed mutant kinases on the phosphorylation of the kinases themselves, however, are unequal, suggesting that Jak1 functions upstream of Tyk2.

The vav proto-oncogene product participates in the signaling pathways activated by various cell-surface receptors, including the type I IFN receptor. During engagement of the type I IFN receptor, p95 vav is phosphorylated on tyrosine residues, but the kinase regulating its phosphorylation has not been identified to date. Our studies demonstrate that p95 vav forms a stable complex with the IFN-receptor-associated Tyk-2 kinase in vivo, and strongly suggest that this kinase regulates its phosphorylation on tyrosine. Thus, p95 vav is engaged in IFN-signaling by a direct interaction with the functional type I IFN receptor complex to transduce downstream signals. © 1997 Federation of European Biochemical Societies.

The goal of this study was to examine follow-up data on the offspring of two parents with NIDDM who have IGT to identify predictors of progression to NIDDM. The study group consisted of 80 individuals with IGT (WHO criteria), 28 of whom had IGT at baseline, and 52 who developed it during follow-up. Both an oral glucose tolerance test (OGTT) and an intravenous glucose tolerance test (IVGTT) were performed at baseline. After a median follow-up interval of 8 years, 35% had progressed to NIDDM, 37% had reverted to normal glucose tolerance, and 29% still had IGT. Those who progressed were younger and more obese than non-progressors. Glucose and insulin values from the OGTT that established the diagnosis of IGT were analysed by multiple logistic regression to determine which time points best discriminated between progressors and non-progressors. High insulin, but not glucose, values at 0 and 120 min distinguished progressors from non-progressors. Values at other sample times that were significantly different (p < 0.05) in progressors were: higher glucose values at 30, 45, and 60 min and higher insulin values at 90 and 180 min. Multiple logistic regression analysis of Bergman's minimal model parameters obtained from the IVGTTs performed at baseline demonstrated that, once IGT is established, low acute phase insulin secretion as well as low insulin sensitivity are significant predictors of progression to NIDDM.

Interferon-alpha (IFN alpha) induces rapid tyrosine phosphorylation of its receptors, two JAK kinases and three STAT transcription factors. One kinase, p135tyk2, is complexed with the IFNaR1 receptor, and may catalyze some of these phosphorylation events. We demonstrate that, in vitro, p135tyk2 phosphorylates two tyrosines on IFNaR1. A phosphopeptide corresponding to the major phosphorylation site (Tyr466) binds STAT2, but not STAT1, in an SH-2-dependent manner. Furthermore, only latent, non-phosphorylated STAT2 interacts with this phosphopeptide. When this phosphopeptide is introduced into permeabilized cells, the IFN alpha-dependent tyrosine phosphorylation of both STATs is blocked. Finally, mutant versions of IFNaR1, in which Tyr466 is changed to phenylalanine, can act in a dominant negative manner to inhibit phosphorylation of STAT2. These observations are consistent with a model in which IFNaR1 mediates the interaction between JAK kinases and the STAT transcription factors.

The denaturing-gradient gel electrophoresis (DGGE) blot technique is a highly sensitive method developed to detect sequence differences (e.g., single base changes and small deletions) in genomic DNA. We used this method to screen for mutations within the p53 locus of independent X-ray-transformed C3H 10T1/2 cell clones. Genomic DNA from 29 transformed clones was digested with Haell and Hinfl endonucleases, electrophoresed on denaturing gradient gels, electroblotted onto nylon membranes, and hybridized to a radioactive p53 cDNA probe. Changes in the melting pattern of p53 were observed in DNAs from 9 of 29 X-ray-transformed clones examined. No changes were found in untransformed, wild-type 10T1/2 cell clones. These results indicate that X-ray-induced p53 mutations may contribute to cell transformation.

Previously, analysis of cDNAs encoding the ltk tyrosine kinase suggested that the structure of this protein was unique among tyrosine kinases, containing a transmembrane domain but only a short, or virtually non-existent, extracellular domain. Further, it was suggested that translational initiation might occur predominantly at a CTG codon. We have now cloned and sequenced a putative full length human ltk cDNA which contains novel sequence information relative to previously identified cDNAs. This ltk cDNA encodes a protein product containing all of the features of typical receptor-type protein tyrosine kinase, including: an ATG translational initiation codon, a secretory signal sequence and a 347 amino acid extracellular domain as well as transmembrane and intracellular kinase domains. Ribonuclease protection analysis indicates that our cloned cDNA represents the most abundant species of mature ltk mRNA. In vitro transcription and translation of the ltk cDNA yields a 100 kDa protein, consistent with initiation at the putative ATG translational codon. In addition, transfection of the ltk cDNA into COS-1 cells produces a similar-sized, glycosylated protein possessing in vitro kinase activity. These data indicate that the ltk gene product likely functions as a cell surface receptor for an unidentified cellular growth factor.